The goal of this research is to determine the mechanism and regulation of the initiation of DNA replication in eukaryotic cells. It is clear that for maintenance of the integrity of the genome from one cell generation to the next, DNA must be duplicated in a highly controlled and accurate manner. Interruption of these controls may promote genome instability and lead to neoplastic transformation. Moreover, the DNA replication proteins represent tangible targets for therapeutic intervention of proliferation of cancer cells, and other proliferative disorders. In recent studies the DNA sequences and initiator protein (ORC) that cooperate to determine the location of origins of DNA replication in Saccharomyces cerevisiae have been identified. The initiator protein is a multi-subunit, sequence-specific DNA binding protein. It interacts with many DNA replication proteins to determine the location of origins of replication within the genome and to control cell cycle specific events that result in genome duplication. The proposed research in this application will investigate how DNA replication occurs in an ORC- and origin-dependent manner and will study how initiation of DNA replication is temporally controlled throughout the cell division cycle. In specific aim 1, the interaction between ORC and Cdc6p will be investigated. In specific aim 2, proteins that function in DNA replication will be reconstituted in vitro to define the mechanism of initiation of DNA replication. In specific aim 3, the cell cycle control of DNA replication and interactions with DNA damage control pathways will be explored. Specific aim 4 has a goal of identifying and characterizing novel ORC binding proteins.